CD40L-binding agent based platelet aggregation assays

ABSTRACT

The present invention provides methods of determining platelet aggregation, methods of determining susceptibility to clotting upon administration of a CD40L-binding moiety, and kits related thereto.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/093,658, filed May 14, 2009 and issued as U.S. Pat. No. 8,409,810,which is the National Stage Application of International Application No.PCT/US2006/044840, filed Nov. 11, 2006, which claims the benefit of U.S.Provisional Application No. 60/737,488, filed Nov. 17, 2005, and U.S.Provisional Application No. 60/804,843, filed Jun. 15, 2006. Thecontents of all of these prior applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention is directed, in part, to methods of determiningplatelet aggregation, methods of determining susceptibility to clottingupon administration of an anti-CD40L antibody, and kits related thereto.

BACKGROUND OF THE INVENTION

Thromboembolic complications (TEC) have been observed in some clinicaltrials of anti-CD40L (CD40L is abbreviated for CD40 ligand, also knownas CD 154) antibody, in particular, humanized monoclonal antibodydirected against CD40L. Known to be expressed by activated T cells,CD40L was also shown to be present on the surface of activated humanplatelets and may play a role in endothelial cell activation in vitro(Henn et al., Nature, 1998, 391, 591-594). However, the role of theCD40/CD40L pathway in regulating coagulation in vivo at the level ofplatelet/endothelial cell interaction is still undefined.

CD40L is a type II membrane protein first described to be expressed onactivated T cells. The interaction of CD40L with its receptor, CD40, iscritical for B cell differentiation, proliferation, and immunoglobulin(Ig) isotype switching induced by helper T cells (Foy et al., Annu. Rev.Immunol., 1996, 14, 591-617; and van Kooten et al., J. Leukoc. Biol.,2000, 67, 2-17). The involvement of the CD40/CD40L pathway in theinteraction of platelets with endothelial cells has recently beendescribed (Henn et al., Nature, 1998, 391, 591-594). Under normalconditions, CD40L is stored in platelets. Upon activation, CD40L istranslocated to the surface of platelets accompanied by surfaceappearance of CD63, P-selectin, and several other proteins, as well asrelease of soluble mediators from intra-platelet granules (Murano G.,Basic Concepts of Hemostasis and Thrombosis, 1980, Boca Raton, Fla., CRCPress, Inc.). Hence, CD40L may play a role in procoagulant activity.

Elevated levels of soluble CD40L (sCD40L) have been found in the bloodof lupus patients (Kato et al., J. Clin. Invest., 1999, 104, 947-955;and Vakkalanka et al., Arthritis Rheum., 1999, 42, 871-881). Rheumatoidfactor (RF), an autoantibody to the Fc portion of Ig, is often detectedin the sera of patients with autoimmune disorders (Mageed et al., Ann.N.Y. Acad. Sci., 1997, 815, 296-311). Additionally, the administrationof anti-CD40L antibody could result in the production of antibodiesdirected against anti-CD40L antibody.

SUMMARY OF THE INVENTION

The present invention provides methods of assaying platelet aggregationcomprising contacting platelets with a platelet activating agent,contacting the activated platelets with a CD40L-binding moiety, andcontacting the activated platelets with a cross-linking agent, whereinaggregation is quantified by sedimentation of platelets. TheCD40L-binding moiety and the cross-linking agent are not a preformedimmune complex. The platelet activating agent is selected from adenosinediphosphate (ADP), collagen, thrombin, thromboxane, neutrophil elastase,p-selectin, and convulxin. The platelets can be obtained from plateletrich plasma (PRP). In some embodiments, the CD40L-binding moiety is ananti-CD40L antibody, such as hu5c8. The cross-linking agent is selectedfrom soluble CD40L (sCD40L), anti-human IgG antibody, anti-hFc antibody,RF, Fc receptor-positive accessory cell, soluble protein A, and solublehuman Fc receptor. The ratio of anti-CD40L antibody to sCD40L can be1:1000 to 1000:1, 1:500 to 500:1, or 3:2.

The present invention also provides methods of assaying plateletaggregation comprising activating platelets by ADP, contacting theactivated platelets with an anti-CD40L antibody, and contacting theactivated platelets with sCD40L or anti-hFc antibody, wherein sedimentedplatelets is indicative of aggregation of the platelets. The anti-CD40Lantibody and sCD40L are not a preformed immune complex.

The present invention also provides methods of determining whether anindividual is susceptible to clotting upon administration of aCD40L-binding moiety comprising removing platelets from the human,contacting the platelets with a platelet activating agent, contactingthe activated platelets with the CD40L-binding moiety, contacting theactivated platelets with a cross-linking agent, and determining thepresence or absence of platelet aggregation, wherein 70% or greaterplatelet aggregation is indicative of susceptibility to clotting. TheCD40L-binding moiety and the cross-linking agent are not a preformedimmune complex. The platelet activating agent is selected from ADP,collagen, thrombin, thromboxane, neutrophil elastase, p-selectin, andconvulxin. The platelets can be obtained from platelet rich plasma(PRP). In some embodiments, the CD40L-binding moiety is an anti-CD40Lantibody, such as hu5c8. The cross-linking agent is selected fromsCD40L, anti-human IgG antibody, anti-hFc antibody, RF, Fcreceptor-positive accessory cell, soluble protein A, and soluble humanFc receptor. The ratio of anti-CD40L antibody to sCD40L can be 1:1000 to1000:1, 1:500 to 500:1, or 3:2.

The present invention also provides methods of determining whether ahuman is susceptible to clotting upon administration of an anti-CD40Lantibody comprising removing platelets from the human, activating theplatelets with ADP, contacting the activated platelets with theanti-CD40L antibody, contacting the activated platelets with sCD4-L oranti-hFc antibody, and determining the presence or absence of plateletaggregation, wherein 70% or greater platelet aggregation is indicativeof susceptibility to clotting. The anti-CD40L antibody and thecross-linking agent are not a preformed immune complex.

The present invention also provides kits comprising a plateletactivating agent, a cross-linking agent, and, optionally, aCD40L-binding moiety. The platelet activating agent is selected fromADP, collagen, thrombin, thromboxane, neutrophil elastase, p-selectin,and convulxin. The CD40L-binding moiety can be an anti-CD40L antibody,such as hu5c8. The cross-linking agent is selected from sCD40L,anti-human IgG antibody, anti-hFc antibody, RF, Fc receptor-positiveaccessory cell, soluble protein A, and soluble human Fc receptor. Thekit may further comprise at least one of a needle, a container foraccepting blood, a container for accepting assay components, andinstructions. In some embodiments, the container for accepting assaycomponents is a cuvette.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Biodata 4-channel platelet aggregation profiler producedtracings depicting the percent of light transmitted through the samplesas compared to the PRP control. The tracings are ended four minutesafter the addition of sub-optimal quantities of ADP, and the final valueconsidered to be the percent of platelets aggregated. An ADP titrationis performed to determine the sub-optimal concentration of ADP for eachPRP sample.

FIG. 2 shows that activated platelets express surface CD40L. Humanplatelets were incubated with or without 10 μM ADP for 1, 10, 20, 40, or60 minutes. As CD40L on the platelet surface is capable of interactingwith biotinylated anti-CD40L antibody, the activation induced increaseof CD40L on the platelet surface is quantified by western blotting forbiotinylated anti-CD40L antibody associated with activated platelets.

FIG. 3 shows that platelet aggregation is affected by complexes ofanti-CD40L antibody and sCD40L. PRP was obtained from ten healthydonors. Anti-CD40L antibody and sCD40L were mixed at least 20 minutesprior to addition to the PRP. A titration was performed for each donorto determine an ADP concentration that produced suboptimal aggregation.Aggregation was induced using the sub-optimal ADP concentration. Eachdot indicates the result from one person.

FIG. 4 shows that platelet aggregation is specifically enhanced bycomplexes of anti-CD40L antibody and recombinant sCD40L. PRP wasobtained from one healthy individual. Aggregation was induced with 0.75μM ADP, which was determined to be suboptimal for this donor. Anti-CD40Lantibody, hIgG, and aglycosylated anti-CD40L antibody were evaluated at200 μg/mL and sCD40L at 30 μg/mL. Anti-CD40L antibody or hIgG was mixedwith recombinant sCD40L for no less than 20 minutes prior to addition tothe PRP-containing cuvette. Bars represent the means and standarddeviations of two data points.

DESCRIPTION OF EMBODIMENTS

The present invention provides methods of assaying platelet aggregation,methods of determining whether a human is susceptible to clotting uponadministration of a CD40L-binding moiety, and to kits that can be usedin these methods.

In one aspect, the present invention provides methods of assayingplatelet aggregation comprising contacting platelets with a plateletactivating agent, contacting the activated platelets with aCD40L-binding moiety, and contacting the activated platelets with across-linking agent. Sedimented platelets is indicative of aggregationof the platelets. The CD40L-binding moiety and the cross-linking agentare not a preformed immune complex when they are added to the platelets.Sedimented platelets is indicative of aggregation of the platelets.

In some embodiments, the platelets are obtained from a mammal such as ahuman. Platelets can be present in PRP obtained from a human.Additionally, the platelets can be isolated from the PRP if so desired.In some embodiments, the platelets or PRP are obtained from a humanprior to administration of a CD40L-binding moiety, such as an anti-CD40Lantibody, to determine, for example, whether the human is at risk ofclotting or platelet aggregation upon administration of theCD40L-binding moiety. Blood can be obtained from humans by widely knowntechniques. PRP can be separated from cellular components by commonlyused separation techniques including, for example, centrifugation andthe like. A typical platelet preparation is described in Example 2below. Purification by Sepharose gel filtration column is described Fineet al., Am. J. Pathol., 1976, 84, 11-24, and in Example 3 below.

The platelets or PRP are contacted by a platelet activating agent. Asused herein, the phrase “platelet activating agent” means any compoundthat can be used to sub-optimally activate a platelet. Sub-optimalactivation of platelets is desired so as to be able to discernaggregation caused by the CD40L-binding moiety. For example, plateletscan be contacted with an amount of a platelet activating agent to inducea threshold, or sub-optimal, amount of platelet aggregation. This amountof platelet aggregation should be less than the amount of aggregationinduced by the CD40L-binding moiety, and may range from 0% aggregationto less than 70% aggregation, or any sub-range therewithin. Plateletactivating agents are well known to the skilled artisan and include, butare not limited to, ADP, collagen, thrombin, thromboxane (TxA2, forexample), human neutrophil elastase (HNE), p-selectin, convulxin, andthe like.

The platelets are also contacted with a CD40L-binding moiety. As usedherein, the phrase “CD40L-binding moiety” is any compound that containsa functional Fc antibody domain and which is also able to bind to CD40L.In some embodiments, the CD40L-binding moiety is an anti-CD40L antibody.As used herein, the phrase “anti-CD40L antibody” means any antibody, orfragment or mutant thereof, that is capable of binding CD40L and/orsCD40L and which is also capable of functionally interacting with the Fcreceptor. Thus, in some embodiments, the anti-CD40L antibody comprises afully intact Fc region. In other embodiments, the anti-CD40L antibodycomprises a fully intact but modified Fc region, wherein themodification does not interfere with Fc receptor binding and/orsignaling. In other embodiments, the anti-CD40L antibody comprises amutant and/or shortened Fc region, wherein the mutation and/orshortening does not interfere with Fc receptor binding and/or signaling.Anti-CD40L antibodies are well known to the skilled artisan and include,for example, the hu5c8. Additional anti-CD40L antibodies include, butare not limited to, M90, M91, M92, IDEC 131, and AnCell anti-CD154 (alsoknown as 24-31), as well as those disclosed in Gray et al., Seminars inImmunol, 1994, 6, 303-310 and Noelle, Immunity, 1996, 4, 415-419. Inaddition, CD40-Fc is a reagents capable of cross-linking CD40L.

The platelets are also contacted with a cross-linking agent. As usedherein, the phrase “cross-linking agent” means any agent that can beused to cross-link CD40L-binding moieties. Suitable cross-linking agentsare well known to the skilled artisan and include, but are not limitedto, sCD40L, anti-human IgG antibodies, anti-hFc antibody, RF, Fcreceptor-positive accessory cells, soluble protein A, and soluble humanFc receptors (such as FcγRI, FcγRII, FcγRIII, and FcRn). The ratio ofCD40L-binding moiety to sCD40L, for example, is 1:1000 to 1000:1, 1:500to 500:1, 1:100 to 100:1, 1:10 to 10:1, or 3:2.

Upon contacting platelets with platelet activating agent, CD40L-bindingmoiety, and a cross-linking agent, sedimentation of platelets isindicative of platelet aggregation. The lack of platelet sedimentationis indicative of a lack of platelet aggregation. In some embodiments, anegative control, or blank, may be used to establish a baseline orthreshold amount of platelet aggregation. A test sample containingplatelets, platelet activating agent, CD40L-binding moiety, andcross-linking agent can be assayed to determine the amount of plateletaggregation, and compared to the baseline platelet aggregation. Anydifference in the amount of platelet aggregation between the test sampleand the negative control can be indicative of platelet aggregation inthe test sample. The assay can be carried out in any suitable containerfor accepting assay components such as, for example, a test tube,microfuge tube, or cuvette.

In another aspect, the present invention also provides methods ofdetermining whether a human is susceptible to clotting uponadministration of a CD40L-binding moiety. As stated above,thromboembolic complications, including clotting, have been observedupon administration of an anti-CD40L antibody to a human. Thus, in someinstances, it is desirable to determine whether a human who iscontemplating receiving an anti-CD40L antibody, or any otherCD40L-binding moiety, is susceptible to establishing such complicationsupon treatment. Representative methods comprise removing platelets fromthe human, activating the platelets with a platelet activating agent asdescribed above, contacting the activated platelets with a CD40L-bindingmoiety as described above, contacting the activated platelets with across-linking agent as described above, and determining the presence orabsence of platelet aggregation. The CD40L-binding moiety and thecross-linking agent are not a preformed immune complex.

Platelet aggregation, and the presence or absence thereof, can bedetermined by numerous means known to those skilled in the art. Forexample, sedimentation of platelets, which is an indication of plateletaggregation, can be monitored as described in the Examples below. Theamount of platelet aggregation can vary from sample to sample dependingupon, for example, the CD40L-binding moiety and the plateletsthemselves. Where the amount of platelet aggregation is 70% or greater,or 75% or greater, or 80% or greater, or 85% or greater, or 90% orgreater, or 95% or greater, it is indicative of susceptibility toclotting and/or thromboembolic complications. A human, who iscontemplating receiving treatment with an anti-CD40L antibody, forexample, and who tests positive (i.e., has greater than 70% plateletaggregation) in the assay method described herein (i.e., the human issusceptible to clotting) may contemplate alternate therapeutic regimens.Indeed, health care professionals may offer alternate therapies uponreceiving a particular result in the assays described herein.

In another aspect, the present invention provides a kit comprising aplatelet activating agent, a cross-linking agent, and, optionally, aCD40L-binding moiety. In some embodiments, the platelet activating agentis selected from ADP, collagen, thrombin, thromboxane, neutrophilelastase, p-selectin, and convulxin. In some embodiments, theCD40L-binding moiety is an anti-CD40L antibody, such as, for example,hu5c8. In some embodiments, the cross-linking agent is selected fromsCD40L, anti-human IgG antibody, anti-hFc antibody, RF, Fcreceptor-positive accessory cell, soluble protein A, and soluble humanFc receptor. The kit can also contain additional items such as, forexample, needle, a container for accepting blood, a container foraccepting assay components, and instructions for carrying out themethods described herein. In some embodiments, the container foraccepting assay components is a cuvette.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner.

EXAMPLES Example 1 Reagents

Research-grade anti-CD40L antibody, recombinant sCD40L, and recombinantsoluble CD40-IgG1 Fc fusion protein (CD40-Fc) were prepared at Biogen(Ferrant et al., Mol. Immunol., 2002, 39, 77-84). The control human IgGwas a human IgG1 kappa purchased from Protos Immunoresearch (SanFrancisco, Calif.). The antibody to the Fc region of human IgG(anti-hFc) was an affinity purified mouse antihuman IgG Fc (JacksonImmunoResearch, West Grove, Pa.). Rb779 is a rabbit antiserum raisedagainst a peptide derived from the C-terminus of CD40L (Garber et al.,J. Biol. Chem., 1999, 274, 33545-33550).

Example 2 Preparation of Platelet Rich Plasma (PRP) and Recovery ofPlatelets

All platelets used in the in vitro platelet aggregation experiments wereisolated from healthy human volunteers who denied ingesting aspirin oraspirin containing compounds within 10 days. Aggregation assays wereperformed on PRP. Approximately 50 mL of whole blood was collected inaliquots in 4.5 mL vacutainer tubes containing 0.5 mL of 3.8% sodiumcitrate. PRP was prepared by centrifuging the anticoagulated blood at200 g for 10 minutes and harvesting the supernatant.

Example 3 Western Blotting

Western blotting for CD40L was performed on purified platelets preparedas following. A total of 50 mL of whole blood was collected in aliquotsin 4.5 mL vacutainer tubes containing 0.5 mL of 3.8% sodium citrate. PRPwas prepared by centrifuging the blood for 20 minutes at 180 g. Thecollected PRP supernatant was loaded onto a Sepharose CL2B (Pharmacia,Peapack, N.J.) gel filtration column, previously equilibrated with twocolumn volumes of phosphate buffered saline (PBS). The platelets wereeluted and washed with PBS. Western blotting was performed to quantitatethe amount of CD40L present in platelets. Briefly, platelets weretreated with Laemmli sample buffer and the proteins resolved byelectrophoresis through a gradient sodium dodecyl sulfate-polyacrylamidegel (Laemmli, Nature, 1970, 227, 680-685). The proteins were transferredto nitrocellulose (Towbin et al., Proc. Natl. Acad. Sci. U.S.A., 1979,76, 4350-4354) and the membrane blotted with Rb779, a rabbit antiserumraised against a peptide derived from the C-terminus of human CD40Lprotein (Garber et al., J. Biol. Chem., 1999, 274, 33545-33550). Thebound antibody was detected by a goat anti-rabbit antiserum conjugatedto horseradish peroxidase (HRP). Serial dilutions of recombinant sCD40Lwere evaluated in parallel with the platelet-derived protein samples.The amount of CD40L in the platelet samples was estimated by comparisonto the recombinant sCD40L standard.

Platelets were incubated with a biotin-conjugated anti-CD40L antibody todetermine whether CD40L is present on the surface of platelets. PRP wasincubated with or without 10 μM ADP for 1, 10, 20, 40, or 60 minutes andthen incubated with biotin-conjugated anti-CD40L antibody. Unboundantibody was removed from the platelets by washing with PBS. The amountof anti-CD40L antibody bound to the platelets was determined by westernblotting using HRP-conjugated streptavidin. The specific binding ofbiotin-conjugated anti-CD40L antibody to the platelets was verified bypreincubating the antibody with recombinant sCD40L.

Example 4 Platelet Aggregation Assay

The Biodata 4-channel platelet aggregation profiler (PAP-4; BiodataCorp., Hatboro, Pa.) was blanked using a cuvette containing onlyplatelet poor plasma (PPP). A 350 μL aliquot of PRP, containingapproximately 2 to 5×10⁸/mL platelets, was added to a cuvette containinga stir bar. Anti-CD40L antibody, human IgG, normal human serum, CD40-Fc,or anti-hFc were added in a total volume of 100 μL. The loaded cuvettewas placed in the machine and the reaction components mixed prior to theaddition of ADP.

Aggregation was initiated with the addition of sub-optimal concentrationof ADP in 50 μL (final concentration varies for each individual sample).The aggregation profiler has four ports, which can run simultaneously.An aggregation tracing was generated for each sample for four minutesfollowing the addition of ADP. At the end of the tracing, the instrumentcalculates the percentage of aggregation by comparing the transmissionof light through the sample to the transmission of light through the PPPblank. A tracing representative of a typical ADP titration is shown inFIG. 1. A titration was performed at the beginning of each experiment,and subsequent runs were performed at a suboptimal ADP concentration.

Example 5 Expression of CD40L on the Surface of Activated Platelets

Expression of CD40L was readily detected in lysates prepared from humanplatelets (data not shown). To determine whether CD40L is expressed onresting and/or activated platelets, platelets were incubated withbiotin-conjugated anti-CD40L antibody and the presence of surface CD40Lwas determined by quantifying the bound biotinylated anti-CD40Lantibody. Surface expression of CD40L was evaluated after 1, 10, 20, 40,and 60 minutes of incubation with or without 10 μM ADP. Surfaceexpression of CD40L was detectable on ADP-activated platelets as earlyas one minute after activation and increased over time (FIG. 2). Thebinding of biotin-conjugated anti-CD40L antibody is specific for CD40L,as preincubation of biotin-conjugated anti-CD40L antibody with sCD40Linhibited binding to activated platelets (data not shown). The amount ofsurface CD40L detected on unactivated (“resting”) platelets alsoincreased over time. This phenomenon is likely attributable to the basallevel of platelet activation under our experimental conditions.

Notwithstanding, the amount of surface CD40L expressed on ADP-treatedplatelets was consistently higher than that of the matched untreatedcontrols. These results are consistent with the notion that CD40L isnormally sequestered inside of platelets and is translocated to thesurface upon activation. Furthermore, the binding of biotin-conjugatedanti-CD40L antibody confirms that anti-CD40L antibody recognizes theCD40L molecules present on the platelet surface.

Example 6 Estimation of the Amount of CD40L in the Platelet Compartment

As CD40L expressed on the surface of activated platelets can berecognized by anti-CD40L antibody, it is important to estimate the sizeof this potential “sink” for anti-CD40L antibody. Using a westernblotting method, the amount of CD40L in platelets was compared to knownamounts of a recombinant sCD40L standard. The amount of CD40L in onemillion platelets is estimated to be approximately 80 pg. Eachmilliliter of freshly drawn blood contains about 300 million platelets,and an average person has a total blood volume of about 5 L. Bycalculation, approximately 120 μg of CD40L is present in the plateletcompartment of an average individual.

Example 7 Effect of Research-Grade Anti-CD40L Antibody on PlateletAggregation

To determine whether the interaction of anti-CD40L antibody with CD40Lexpressed on activated platelets influences the hemostatic cascade, theaggregation of healthy human platelets in the presence of research-gradeanti-CD40L antibody was examined. Research-grade anti-CD40L antibody didnot affect the aggregation of ADP-activated platelets over a wide rangeof ADP concentrations (Tables 1 through 3). Resting platelets were alsonot affected by anti-CD40L antibody (Tables 1 and 3-5). As expected,control human IgG antibody (Tables 2 and 3) did not affect plateletaggregation. CD40-Fc was evaluated to determine whether the binding of adimeric receptor protein to the CD40L expressed on the surface ofplatelets would influence aggregation. CD40-Fc did not affect plateletaggregation (Table 4).

Example 8 Effect of Cross-linked Anti-CD40L Antibody on PlateletAggregation

Anti-CD40L Antibody Cross-linked by Antibodies

To determine if there is an effect on aggregation when anti-CD40Lantibody is cross-linked by an antibody specific for the Fc region ofIgG, activated and resting platelets were treated with anti-CD40Lantibody with or without anti-hFc as a cross-linking agent. Oneexperiment was performed in which the effects of cross-linked anti-CD40Lantibody were cross-titrated with varied amounts of ADP and anti-hFc(Table 5). Cross-linking of anti-CD40L antibody with 20 μg/mL or 6.66μg/mL anti-hFc did not affect platelet aggregation (Table 5). However,increased aggregation was observed when anti-CD40L antibody wascross-linked with 2.22 μg/mL of anti-hFc (Table 5 compare lines 109 and110). Such an enhancing effect was not observed when a lowerconcentration of ADP was used for activation (Table 5). Platelets werenot affected by anti-CD40L antibody in the presence of normal humanserum, which contains antibodies that are not expected to cross-linkanti-CD40L antibody (Table 6). These results suggest that enhancement ofplatelet aggregation by anti-CD40L antibody cross-linked by antibodiesis possible.

Anti-CD40L Antibody Cross-linked by Soluble CD40 Ligand

Multiple concentrations of anti-CD40L antibody and sCD40L were evaluatedin the platelet aggregation assay to determine whether anti-CD40Lantibody cross-linked by sCD40L would enhance platelet aggregation.Based on the number of binding sites available on sCD40L and anti-CD40Lantibody, anti-CD40L antibody would be maximally cross-linked when twomolecules of trimeric sCD40L are bound to three molecules of divalentanti-CD40L antibody. By calculation, the weight/weight ratio for maximalcross-linking is theoretically 3.75 anti-CD40L antibody to 1 sCD40L.Several ratios in this range were tested, and 233 μg/mL anti-CD40Lantibody and 30 μg/mL sCD40L was empirically found to be a condition formaximal cross-linking of anti-CD40L antibody. Ten-fold lower andten-fold higher concentrations of anti-CD40L antibody were alsoevaluated with 30 μg/mL sCD40L. This test has been done using PRPprepared from ten healthy donors. Interestingly, enhanced aggregationoccurred with platelets isolated from some, but not all, healthy donors.

Platelet aggregation was induced by anti-CD40L antibody maximallycross-linked by sCD40L (FIG. 3). An aglycosylated version of anti-CD40Lantibody cross-linked by sCD40L did not enhance platelet aggregation,indicating the effect is FcγRIIa-dependent (FIG. 4). Control human IgG(hIgG) and sCD40L together had no effect on platelet aggregation (FIG.4).

TABLE 1 Effect of anti-CD40L antibody on ADP-Activated Platelets Sample# BG9588 ADP Concentration (μM) % of Aggregation 1 20.00 73 2 + 20.00 723 6.70 60 4 + 6.70 71 5 2.00 25 6 + 2.00 21 7 0.70 11 8 + 0.70 24 9 0.3712 10 + 0.37 15 11 0.00 3 12 + 0.00 11

Anti-CD40L antibody does not induce the aggregation of resting plateletsor affect the aggregation of ADP-activated platelets. PRP was placed ina cuvette and stirred with or without 5 μg/mL research-grade anti-CD40Lantibody. ADP was added to the final concentrations listed. Afour-minute aggregation tracing was generated for each sample and thepercent of aggregation calculated.

TABLE 2 Effect of anti-CD40L antibody and IgG on ADP-Activated PlateletsSample # BG9588 Human IgG ADP (μM) % of Aggregation 13 20.00 77 14 +20.00 89 15 + 20.00 80 16 6.70 74 17 + 6.70 75 18 + 6.70 78 19 2.00 1020 + 2.00 12 21 + 2.00 12 22 0.70 8 23 + 0.70 11 24 + 0.70 10 25 0.37 926 + 0.37 10 27 + 0.37 10 28 0.00 3

Anti-CD40L antibody and a control human IgG do not affect theaggregation of ADP-activated platelets. PRP was placed in a cuvette andstirred with or without 5 μg/mL of research-grade anti-CD40L antibody orhuman IgG. ADP was added to the final concentrations listed. Afour-minute aggregation tracing was generated for each sample and thepercent of aggregation calculated.

TABLE 3 Effect of anti-CD40L antibody and IgG on ADP-Activated PlateletsSample # BG9588 Human IgG ADP (μM) % of Aggregation 29 0.00 1 30 + 0.003 31 + 0.00 3 32 20.00 70 33 + 20.00 68 34 + 20.00 68 35 10.00 64 36 +10.00 69 37 + 10.00 69 38 5.00 72 39 + 5.00 70 40 + 5.00 67 41 2.50 6542 + 2.50 57 43 + 2.50 62 44 1.25 13 45 + 1.25 15 46 + 1.25 12 47 0.6010 48 + 0.60 9 49 + 0.60 10 50 0.30 10 51 + 0.30 10 52 + 0.30 10

Anti-CD40L antibody and a nonspecific human IgG do not induce theaggregation of resting platelets or affect the aggregation ofADP-activated platelets. PRP was placed in a cuvette and stirred with orwithout 5 μg/mL of research-grade anti-CD40L antibody or human IgG. ADPwas added to the final concentrations listed. A four-minute aggregationtracing was generated for each sample and the percent of aggregationcalculated.

TABLE 4 Effect of anti-CD40L antibody and CD40-Fc on ADP-ActivatedPlatelets BG9588 CD40Ig ADP % of Sample # (μg/mL) (μg/mL) (μM)Aggregation 53 0.00 2 54 20.00 0.00 3 55 10.00 0.00 3 56 5.00 0.00 3 572.50 0.00 5 58 1.25 0.00 3 59 20.00 0.00 3 60 10.00 0.00 3 61 5.00 0.003 62 2.50 0.00 3 63 1.25 0.00 3 64 0.63 0.00 3 65 20.00 84 66 5.00 79 6710.00 5.00 79 68 2.50 77 69 10.00 2.50 81 70 1.25 61 71 10.00 1.25 64 720.60 15 73 10.00 0.60 16

The binding of anti-CD40L antibody or CD40-Fc to CD40L on the surface ofactivated platelets does not affect platelet aggregation. PRP was placedin a cuvette and stirred with or without research-grade anti-CD40Lantibody or CD40-Fc at the concentrations listed. ADP was added to thefinal concentrations listed. A four-minute aggregation tracing wasgenerated for each sample and the percent of aggregation calculated.

TABLE 5 Effect of Cross-linked anti-CD40L antibody on ADP-ActivatedPlatelets Sample # BG9588 Anti-hFc (μg/mL) ADP (μM) % of Aggregation 980.0 2 99 20.0 76 100 20.00 0.0 3 101 + 0.0 3 102 + 20.00 0.0 3 103 20.001.0 73 104 + 20.00 1.0 83 105 + 6.66 0.0 3 106 6.66 1.0 80 107 + 6.661.0 84 108 + 2.22 0.0 3 109 2.22 1.0 23 110 + 2.22 1.0 76 111 + 20.000.0 4 112 20.00 0.5 6 113 + 20.00 0.5 8 114 + 6.66 0.0 3 115 6.66 0.5 8116 + 6.66 0.5 8 117 + 2.22 0.0 3 118 2.22 0.5 7 119 + 2.22 0.5 8

The cross-linking of anti-CD40L antibody bound to CD40L on the surfaceof activated platelets may enhance aggregation under limited conditions.PRP was placed in a cuvette and stirred with or without research-gradeanti-CD40L antibody at 2 μg/mL and/or anti-hFc at the concentrationslisted. ADP was added to the final concentrations listed. A four-minuteaggregation tracing was generated for each sample and the percent ofaggregation calculated.

TABLE 6 Effect of Normal Human Serum on Resting Platelets Sample #BG9588 Serum (μg/mL) % of Aggregation 74 2 75 1.0x 8 76 0.2x 7 77 0.04x5 78 0.008x 5 79 0.0016x 6 80 0.00032x 6 81 + 1.0x 9 82 + 0.2x 6 83 +0.04x 6 84 + 0.008x 7 85 + 0.0016x 6 86 + 0.00032x 6

Normal human serum does not affect the aggregation of resting platelets.PRP was placed in a cuvette and stirred with or without research-gradeanti-CD40L antibody at 2 μg/mL and/or normal human serum at theconcentrations listed. A four-minute aggregation tracing was generatedfor each sample and the percent of aggregation calculated.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

What is claimed is:
 1. An in vitro method of assaying plateletaggregation comprising: providing a sample comprising platelets obtainedfrom a human subject; contacting the sample with a CD40L-binding moiety;contacting the sample with a cross-linking agent capable ofcross-linking the CD40L binding moiety; contacting the sample with aplatelet activating agent; and determining the amount of plateletaggregation in the sample, wherein sedimented platelets is indicative ofaggregation of the platelets.
 2. The method of claim 1, wherein theCD40L-binding moiety is an antibody or an antigen-binding fragmentthereof.
 3. The method of claim 1, wherein the cross-linking agent isselected from the group consisting of sCD40L, anti-human IgG antibody,anti-hFc antibody, rheumatoid factor (RF), Fc receptor-positiveaccessory cell, soluble protein A, and soluble human Fc receptor.
 4. Themethod of claim 1, wherein the platelet activating agent is in an amountsuitable to induce a sub-optimal amount of platelet aggregation in thesample.
 5. The method of claim 4, wherein the sub-optimal amount ofplatelet aggregation is less than 70% platelet aggregation.
 6. Themethod of claim 4, wherein a titration is first performed on theplatelets to determine the amount of platelet activating agent suitableto induce a sub-optimal amount of platelet aggregation in the sample. 7.The method of claim 1, wherein the platelet activating agent is selectedfrom the group consisting of ADP, collagen, thrombin, thromboxane,neutrophil elastase, p-selectin and convulxin.
 8. The method of claim 7,wherein the platelet activating agent is ADP and wherein the ADP ispresent in the sample at a final concentration of 1 μM.
 9. The method ofclaim 2, wherein the ratio of anti-CD40L antibody or antigen-bindingfragment thereof to cross-linking agent is 1:1000 to 1000:1, 1:500 to500:1 or 3:2.
 10. The method of claim 1, wherein the sample comprisingplatelets is obtained from platelet rich plasma.
 11. The method of claim1, wherein the CD40L-binding moiety and the cross-linking agent aremixed prior to addition to the sample comprising platelets.
 12. Themethod of claim 11, wherein the CD40L-binding moiety and thecross-linking agent are mixed for no less than 20 minutes prior toaddition to the sample comprising platelets.
 13. The method of claim 1,wherein the CD40L-binding moiety and the cross-linking agent are not apreformed immune complex.
 14. The method of claim 1, wherein the amountof platelet aggregation in the sample is compared to the amount ofplatelet aggregation in a control sample.
 15. The method of claim 14,wherein the control sample is contacted with an anti-CD40L antibody orantigen-binding fragment thereof or with a control human IgG.
 16. Themethod of claim 2, wherein the anti-CD40L antibody or antigen-bindingfragment thereof is hu5c8 or an antigen-binding fragment thereof.
 17. Anin vitro method of assaying platelet aggregation comprising: providing asample comprising platelets obtained from a human subject; contactingthe sample with an hu5c8 antibody or antigen binding fragment thereof;contacting the sample with a cross-linking agent capable ofcross-linking the hu5c8 antibody or antigen-binding fragment thereof;wherein the cross-linking agent is selected from the group consisting ofsCD40L and anti-hFc antibody; contacting the sample with a ADP;determining the amount of platelet aggregation in the sample; whereinsedimented platelets is indicative of aggregation of the platelets. 18.The method of claim 17, wherein the ADP is in an amount suitable toinduce a sub-optimal amount of platelet aggregation in the sample. 19.The method of claim 18, wherein the sub-optimal amount of plateletaggregation is less than 70% platelet aggregation.
 20. The method ofclaim 18, wherein a titration is first performed on the platelets todetermine the amount of ADP suitable to induce a sub-optimal amount ofplatelet aggregation in the sample.
 21. The method of claim 17, whereinthe ADP is present in the sample at a final concentration of 1 μM. 22.The method of claim 17, wherein the ratio of hu5c8 antibody orantigen-binding fragment thereof to cross-linking agent is 1:1000 to100:1, 1:500 to 500:1, or 3:2.
 23. The method of claim 17, wherein thesample comprising platelets is obtained from platelet rich plasma. 24.The method of claim 17, wherein the hu5c8 antibody or antigen bindingfragment thereof and the cross-linking agent are mixed prior to additionto the sample comprising platelets.
 25. The method of claim 24, whereinthe hu5c8 antibody or antigen binding fragment thereof and thecross-linking agent are mixed for no less than 20 minutes prior toaddition to the sample comprising platelets.
 26. The method of claim 17,wherein the hu5c8 antibody or antigen binding fragment thereof and thecross-linking agent are not a performed immune complex.
 27. The methodof claim 17, wherein the amount of platelet aggregation in the sample iscompared to the amount of platelet aggregation in a control sample. 28.The method of claim 27, wherein the control sample is contacted with anaglycosylated hu5c8 antibody or antigen-binding fragment thereof or witha control human IgG.